Genome-wide analysis of the peanut CaM/CML gene family reveals that the AhCML69 gene is associated with resistance to Ralstonia solanacearum

Background Calmodulins (CaMs)/CaM-like proteins (CMLs) are crucial Ca2+-binding sensors that can decode and transduce Ca2+ signals during plant development and in response to various stimuli. The CaM/CML gene family has been characterized in many plant species, but this family has not yet been characterized and analyzed in peanut, especially for its functions in response to Ralstonia solanacearum. In this study, we performed a genome-wide analysis to analyze the CaM/CML genes and their functions in resistance to R. solanacearum. Results Here, 67, 72, and 214 CaM/CML genes were identified from Arachis duranensis, Arachis ipaensis, and Arachis hypogaea, respectively. The genes were divided into nine subgroups (Groups I-IX) with relatively conserved exon‒intron structures and motif compositions. Gene duplication, which included whole-genome duplication, tandem repeats, scattered repeats, and unconnected repeats, produced approximately 81 pairs of homologous genes in the AhCaM/CML gene family. Allopolyploidization was the main reason for the greater number of AhCaM/CML members. The nonsynonymous (Ka) versus synonymous (Ks) substitution rates (less than 1.0) suggested that all homologous pairs underwent intensive purifying selection pressure during evolution. AhCML69 was constitutively expressed in different tissues of peanut plants and was involved in the response to R. solanacearum infection. The AhCML69 protein was localized in the cytoplasm and nucleus. Transient overexpression of AhCML69 in tobacco leaves increased resistance to R. solanacearum infection and induced the expression of defense-related genes, suggesting that AhCML69 is a positive regulator of disease resistance. Conclusions This study provides the first comprehensive analysis of the AhCaM/CML gene family and potential genetic resources for the molecular design and breeding of peanut bacterial wilt resistance. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-024-10108-5.


Background
Plants are faced with various stresses throughout their lifetime.To overcome these challenges, plants perceive and translate these external stimuli into an internal response via complex signaling networks.Calcium (Ca 2+ ), a second messenger of signal transduction in plant cells, plays important roles in plant growth and development as well as in coping with stress [1].When plants experience external stimulation, the concentration of cytosolic free Ca 2+ increases, which activates a calcium signature [2].These Ca 2+ signatures are decoded and transmitted into downstream responses by a toolkit of calcium-binding proteins, which are referred to as calcium sensors [3].The major sensors, e.g., calmodulins (CaMs), CaM-like proteins (CMLs), Ca 2+ -dependent protein kinases (CDPKs), and calcineurin B-like proteins (CBLs), usually contain a number of EF-hand motifs [4,5].Each EF-hand motif consists of 29 amino acid residues forming two α-helices that are connected by a 12-amino acid loop [6].The Asp (D) and Glu (E) amino acids in the EF-hand motif are conserved and constitute the D-x-D or D/E-E-L motif, which can bind to Ca 2+ [7,8].An EF-hand binds one Ca 2+ ion, inducing a conformational change in the Ca 2+ sensor protein, interacting with downstream proteins or regulating their catalytic activity [9,10].
CaMs, which have four EF-hand motifs, are conserved Ca 2+ sensors in both plants and animals.CMLs, normally with 1-6 EF-hand motifs, display some sequence homology with CaM and exhibit structural differentiation in plants [11,12].Genome-wide analysis of CaM/CML genes has been performed for many plant species, including Arabidopsis (7 CaMs and 50 CMLs), rice (Oryza sativa, 5 CaMs and 32 CMLs), Brassica napus (25 CaMs and 168 CMLs), tomato (Solanum lycopersicum, 6 CaMs and 52 CMLs), and wheat (Triticum aestivum, 18 CaMs and 230 CMLs) [13,14].Although CMLs are structurally homologous to CaMs, plants have far more CMLs than CaMs.CaM/CMLs are widely involved in plant growth and development, as well as in the response to various environmental stimuli [15].In Arabidopsis, CaM3 activates the shock transcription factor (HSF) by regulating the activity of CaM-binding protein phosphatase (PP7) or protein kinase (CBK3), resulting in heat resistance [16,17].CMLs have diverse functions in the plant immune response, and AtCML8, AtCML9, and AtCML24 function as positive regulators in response to Pseudomonas syringae pv.Tomato DC3000, while AtCML46 and AtCML47 have been demonstrated to be negative monitors [18,19].CMLs can be regulated by transcription factors (MYB and bZIP) or miRNAs to form a feedback loop to confer pathogen resistance.In upland cotton, GhMYB108 interacts with GhCML11 and forms a positive feedback regulatory loop to participate in resistance to Verticillium dahlia infection [20].Pepper CaCML13 is positively regulated by CabZIP63 at the transcriptional level and forms a positive feedback network to prevent R. solanacearum infection (RSI) [21].In rice, overexpression of OsCaML2 reduces resistance to Xanthomonas oryzae pv.Oryzae (Xoo) [22].OsCaML2 was demonstrated to be the target protein of osa-miR1432, and the highly inducible osa-miR1432 suppressed the expression of OsCaML2 to increase disease resistance.The CML-mediated network can induce changes in downstream hormone levels, including changes in salicylic acid (SA) and jasmonic acid (JA) signaling.In tomato, silencing SlCML55 increased PR1 expression and stimulated the SA immune response, ultimately resulting in resistance to Phytophthora capsica [23].Arabidopsis AtCML37 and AtCML42 have been demonstrated to play roles in defense against herbivory and some pathogen attacks, which is related to calcium and JA signaling [24][25][26].Although the function of CaMs/CMLs in response to various stimuli has been systematically studied in several plant species, genomewide analysis of the CaM/CML gene families of peanut has rarely been performed, and the possible functions of peanut CaM/CML genes are still unclear.
Cultivated peanut is an important oil and economic crop in tropical and subtropical regions of the world.Bacterial wilt (BW) is a destructive soilborne disease caused by R. solanacearum that can cause severe problems in peanut yield and quality.More than 200 plant species from 54 families can be infected by R. solanacearum, resulting in very serious economic losses worldwide every year [27].Such losses may cause more than 10% decreases in peanut production or even result in the death of the entire crop.Although many disease resistance (R) genes and resistance-related genes have been found in plants, the R genes for BW are still poorly understood [28].Two R genes, RRS1-R/RPS4 and ERECTA , act as positive regulators of resistance to BW and have been extensively studied in A. thaliana [29].Only three BW resistance-related genes, AhRLK1, AhRRS5, and AhGLK1b, have been cloned from peanut [30][31][32].Separately overexpressing the three genes in tobacco caused resistance to R. solanacearum.As a result, additional studies are necessary to identify BW resistance (or related) genes in peanut plants, which will be beneficial for revealing the molecular pathways involved in peanut resistance to R. solanacearum and accelerating the breeding of peanut plants resistant to BW.
In the present study, we first performed a genome-wide analysis to identify CaM/CML genes in A. duranensis, A. ipaensis and A. hypogaea.The characteristics of the CaM/CML genes, including intron-exon organization, chromosomal location, EF-hand motifs, and phylogenetic relationships, were evaluated.To demonstrate the involvement of the CaM/CML genes in peanut responses to R. solanacearum, the expression patterns of these genes were also analyzed through RNA-seq data from peanut leaves with RSI [33].The function of AhCML69 was further investigated due to its significantly upregulated expression following R. solanacearum infection.Transient overexpression of AhCML69 increased the resistance to R. solanacearum in Nicotiana benthamiana.Our results will be useful for understanding the functions of AhCaM/CML in modulating peanut responses to R. solanacearum.

Identification and characterization of CaM/CML genes
Through a BLASTP search of Arabidopsis AtCaM/CML protein sequences, 67, 72, and 214 CaM/CML genes were identified in A. duranensis (AdCaMs/CMLs), A. ipaensis (AiCaMs/CMLs), and A. hypogaea (allotetraploid peanut cultivar; AhCaMs/CMLs), respectively.All the members were submitted to InterPro and SMART to verify the presence of the EF-hand motif domain.All the CaM proteins and 70 AhCML, 13 AdCML and 10 AiCML proteins had four EF-hand motifs.The remaining CML proteins had varying numbers of EF-hand motifs (1, 2, 3, 4 or 6) (Additional File 1).Detailed information for all the identified CaM/CML genes, including gene ID, chromosome location, molecular weight (MW), isoelectric point (PI), and number of EF-hand motifs, is provided in Additional File 1.
The gene structures of the CaM/CML members were analyzed according to their exon-intron organization.Most of the CaM/CML genes had more than one intron (Fig. S1D-F).No introns were found in 77 (35.98%)AhCaM/CML genes, while more than one intron was identified in the other genes (Fig. S1F).In the two diploid species, the CaM/CML gene structures were generally similar.There were 12 (17.65%)AdCaM/CML genes with no introns, and the others contained 3-5 introns in A. duranensis (Fig. S1D).The number of introns varied from 0 to 11 in A. ipaensis.There were 14 (19.44%)AiCaM/CML genes without introns, and the others had 1-11 introns (Fig. S1E).

Phylogenetic and conserved motifs of CaM/CML proteins
To evaluate the evolutionary relationships of the CaM/ CML gene family, we constructed a phylogenetic tree by using the NJ method (1000 bootstrap replicates) to associate the AdCaM/CML, AiCaM/CML, and AhCaM/ CML proteins with the Arabidopsis AtCaM/CML proteins.These CaM/CML proteins were divided into nine subgroups (Fig. 1A  By analyzing the domains and motifs of the identified CaM/CML proteins, six additional motifs were predicted via the MEME website (Fig. 1B and Additional File 4).Motif 1 includes the core conserved sequence (D-x-D) in the EF-hand motif, and motif 2 contains a helix-loophelix sequence.All CaM/CML proteins consist of one to six copies of motif 1.All Group I, II (CaMs) and IV proteins had four copies of motif 1.The other group of CML proteins included motifs 2-5 (Fig. 1C and Additional Files 3 and 4).Sequence analysis of AhCaM/CML proteins demonstrated that both AhCaMs and AhCMLs contain EF-hand motifs, while the sequence similarity of CaMs with EF-hand motifs was greater than that of CMLs.

Gene duplication and collinearity analysis of the CaM/CML genes
To elucidate the role of gene duplication in the evolution of the AhCaM/CML genes, the duplication events of these genes were investigated via collinearity analysis.A total of 81 pairs of homologous AhCaM/CML genes were found in peanut (Fig. 2A and Additional File 5).Further analysis revealed that 214 AhCaM/CML genes were derived from gene duplication, of which 110 genes were derived from whole-genome duplication, 71 from tandem repeats, and 33 from scattered repeats (Additional File 6).These findings indicated that the expansion of the AhCaM/CMLs occurred mainly through genome-wide duplication.In addition, we investigated the synteny relationships of the CaM/ CML genes between peanut and the two wild species.Sixty-seven pairs of homologous CaM/CML genes were identified between peanut and A. duranensis, and 72 pairs of homologous CaM/CML genes were identified between peanut and A. ipaensis (Fig. 2B and Additional File 7).These results suggested that the allotetraploid peanut evolved from these two wild species.To explore the evolutionary selection pressure on AhCaM/CML genes, the Ka/Ks values of the AhCaM/CML gene pairs were calculated using TBtools software.The results showed that the Ka/Ks ratios of all ortholog pairs were less than 1.0 (Additional File 8), indicating that the homologous genes had undergone intensive purifying selection pressure and remained conserved in both structure and function in peanut.

Expression profile analysis of AhCaM/CML genes in response to R. solanacearum
To explore the potential functions of the AhCaM/CML genes in response to R. solanacearum infection, we analyzed the expression patterns of these genes in resistant and susceptible peanuts after R. solanacearum infection based on previous transcriptome data [33].Two AhCML genes, AhCML69 (AH08G06260.1)and AhCML174 (AH17G29650.1),were selected as candidate genes due to their high expression after R. solanacearum infection (Fig. 3B).The spatial and temporal expression profiles of the two AhCML genes were further investigated based on publicly available transcriptome datasets [34].These genes were highly expressed in the roots, stems, leaves, and flowers (Fig. 3A), indicating that they may have extensive functions.To verify the consistency of the expression pattern, the expression of AhCML69 in different tissues of peanut plants was determined via qRT-PCR in three biological replicates.These results were in substantial agreement with the previous transcriptome analysis results, which revealed that the gene was highly expressed in the roots, stems, leaves, and flowers (Fig. 3C).In addition, the expression of AhCML69 and AhCML174 increased in response to low temperature or salicylic acid treatment (Fig. 3A), suggesting that these genes are likely involved in the response to environmental stimuli.

Cloning and functional validation of candidate AhCML genes
The two AhCML genes AhCML69 and AhCML174 were successfully cloned from the leaf cDNA of resistant peanut plants infected with R. solanacearum.The ORF lengths were 488 and 908 bp, encoding 163 and 303 amino acids, respectively (Additional File 9).To analyze the functions of these genes in plants, transient overexpression vectors encoding AhCML69 and AhCML174 were constructed and subsequently transformed into the leaves of N. benthamiana via Agrobacterium transformation (Fig. 4A).Compared with that in control leaves agroinfiltrated with 35S::00, leaf necrosis was obvious at 48 hours post-agroinfiltration in the AhCML69-and AhCML174-overexpressing leaves, although necrosis was less evident in the AhCML174-overexpressing leaves (Fig. 4B).These phenotypic results demonstrated that AhCML69 and AhCML174 might play a certain role in mediating cell death to varying degrees.

Sequence analysis and subcellular localization of the AhCML69 protein
The AhCML69 gene encodes a calmodulin-like protein with four EF-hand motifs and is located on chromosome 8 (Additional File 1).This protein consists of 163 amino acids; and has a MW of 18746.22 and a pI of 4.17 (Additional File 1).To further evaluate the sequence similarity between AhCML69 and other CMLs, BLASTP analysis was performed by using the full-length amino acid sequence from the NCBI website, and the results suggested that AhCML69 was closely related to Arabidopsis AtCML44, rice OsCML44 and pepper CaCML44.
Multiple sequence alignment and phylogenetic analysis also verified that AhCML69 was highly similar to AtCML44, OsCML44, and CaCML44 (Fig. 5A, B).

Transient overexpression of AhCML69 causes cell death in N. benthamiana leaves
To further demonstrate that AhCML69 is involved in disease resistance, the recombinant plasmid 35S::AhCML69 and the control 35S::00 were transiently overexpressed in N. benthamiana leaves by Agrobacterium transformation.AhCML69 was highly expressed at 48, 72, and 96 hpi in the 35S::AhCML69 leaves of N. benthamiana (Fig. 6B).Compared with that in 35S::00 plants, cell death was more severe in the leaves of 35S::AhCML69 plants at 48 hpi, which was also verified by trypan blue staining and electrolyte leakage measurements (Fig. 6AC).In the trypan blue staining experiment, the color of the 35S::AhCML69 leaves was deeper blue than that of the control leaves at 48 hpi, and the electrolyte leakage of the 35S::AhCML69 leaves was significantly greater than that of the control leaves at 48, 72, and 96 hpi.These results suggested that there was severe cell death in the leaves of 35S::AhCML69.DAB (3,3'-diaminobenzidine) staining revealed significantly greater brown staining in the 35S::AhCML69overexpressing leaves than in the control leaves at 48 hpi, indicating that AhCML69 increased the content of active oxygen species and was likely involved in the plant defense response.

Transient overexpression of AhCML69 positively mediates the defense response
To confirm the resistance effect of AhCML69, the expression levels of immune-related marker genes were determined in 35S::AhCML69 and 35S::00 leaves at 48, 72 and 96 hpi by qRT-PCR (Fig. 7).Compared with those in 35S::00 leaves, in 35S::AhCML69 leaves, the expression of the hypersensitive response (HR) marker genes NbH1N1 and NbHsr203J was significantly upregulated at 72 and 96 hpi, indicating that cell death was associated with HR (Fig. 7A).The PTI (PAMP-triggered immunity)-related genes NbWIPK at 48 and 96 hpi and NbPTI5 at 48 hpi were upregulated in 35S::AhCML69 leaves (Fig. 7B).These results suggested that AhCML69 positively regulates the PTI response.The JA signaling pathway-related genes NbOPR3 at 48 hpi and NbLOX at 72 hpi and the SA signaling pathway-related genes NbPR1 and NbPR2 at all timepoints were upregulated in 35S::AhCML69 leaves (Fig. 7C, D).These results suggested that AhCML69 positively regulates the defense response by regulating the JA and SA signaling pathways.

Transient overexpression of AhCML69 enhanced resistance to R. solanacearum
To determine the function of AhCML69 in resistance to R. solanacearum, we transiently overexpressed 35S::AhCML69 and 35S::00 in N. benthamiana leaves and subsequently inoculated them with R. solanacearum.qRT-PCR in 35S::AhCML69 tobacco leaves at 24 and 48 hpi revealed high expression of AhCML69 (Fig. 8B), indicating its successful expression.Disease symptoms and the expression levels of immunerelated marker genes were subsequently evaluated in tobacco leaves.Compared with that in 35S::00 plants, cell death in 35S::AhCML69 tobacco leaves was lower after R. solanacearum inoculation (Fig. 8A), which was also further demonstrated by electrolyte leakage measurements (Fig. 8C).These findings suggested that cell death, a disease symptom caused by R. solanacearum infection, was retarded to a certain degree.The HR marker genes NbH1N1 and NbHsr203J at 24 and 48 hours post-inoculation with R. solanacearum (hpir), the PTI-related gene NbWIPK at 24 and 48 hpir, and NbPTI5 at 24 hpir were upregulated in 35S::AhCML69 leaves (Fig. 8D, E).The JA signaling pathway-related genes NbOPR3 at 24 and 48 hpir and NbLOX at 24 hpir and the SA signaling pathway-related genes NbPR1 and NbPR2 at 24 hpir were upregulated in 35S::AhCML69 leaves (Fig. 8F, G).These results suggested that AhCML69 enhances tobacco resistance to

Discussion
CaM is a conserved protein found in both plants and animals, while CML is a unique protein found in plants.The CaM/CML gene family has been demonstrated to play important roles in plant development and the response to various stimuli [15].However, there have been no genome-wide analyses of the CaM/CML gene family in Arachis species.In the present study, the distribution and structural and functional characteristics of the CaM/ CML genes in A. hypogaea were investigated, which could provide a comprehensive understanding of the evolutionary history and functional roles of this gene family.Twenty-three CaM and 191 CML members were identified in peanut, which is more than in most plant species, including A. duranensis (9 CaM and 58 CML), A. ipaensis (14 CaM and 58 CML), Arabidopsis (7 CaMs and 50 CMLs), rice (5 CaMs and 32 CMLs), tomato (6 CaMs and 52 CMLs), and B. napus (25 CaMs and 168 CMLs) [14].This is likely because peanut (AABB, 2n = 40) was formed by natural allopolyploidization between A. duranensis (AA, 2n = 20) and A. ipaensis (BB, 2n = 20), and its genome (2.38 Gb) is larger than that of the other plants.Collinearity analysis revealed that almost all the CaM/ CML genes in the two wild species were homologous to genes in peanut (Fig. 2B and Additional File 7), indicating that the allotetraploid peanut evolved from these two diploid species.Gene duplication, often derived from polyploidization (mainly segmental, whole-genome or tandem duplication), plays important roles in gene family expansion [35,36].There were 81 pairs of homologous of CaM/CML genes in peanut (Fig. 2A and Additional File 5).Among the AhCaM/CML genes, whole-genome duplication, tandem repeats, and scattered repeats were the most common duplication types (Additional File 6).To illustrate the evolutionary limitations and selection pressures on the AhCaM/CML genes, Ka, Ks, and Ka/ Ks values for 81 homologous AhCaM/CML gene pairs were calculated (Additional File 8).Ks values between gene pairs, indicating the rate of background base substitution, can be used to estimate the time since wholegenome duplication [37].The Ks values for the AhCaM/ CML gene pairs varied from 0.007 to 2.08, suggesting that a large-scale AhCaM/CML gene duplication event occurred (Additional File 8).The Ka/Ks ratios of all ortholog pairs were less than 1.0 in peanut (Additional The spatiotemporal expression patterns of the AhCaM/ CML genes may be associated with their functions.Our previous study showed that many Ca 2+ sensor genes are significantly induced by RSI in peanut leaves [33].The potential relationship between the expression levels of the AhCaM/CML genes and RIS was investigated, and the expression levels of AhCML69 and AhCML174 were found to be positively correlated with RIS in peanut leaves (Fig. 3B).Tissue expression analysis demonstrated that AhCML69 and AhCML174 were highly expressed in roots, stems, leaves, and flowers (Fig. 3AC).In addition, AhCML69 and AhCML174 were induced by low-temperature or salicylic acid treatment (Fig. 3A).In Arabidopsis, AtCML8 is highly expressed in roots, leaves, and flowers and is involved in resistance to R. solanacearum [38].CaCML13 is significantly upregulated by RIS in pepper roots, where R. solanacearum invades, and plays an important role in pepper immunity against R. solanacearum [21].TaCML36 is significantly induced by the soil-borne fungus Rhizoctonia cerealis in resistant wheat stems where disease symptoms appear and positively participates in the immune response to R. cerealis [39].Plant roots, especially the lateral roots and tips, are the main sites where R. solanacearum invades the host [40].Once R. solanacearum enters plant roots, it invades xylem vessels to spread toward the aerial parts of the host through the vascular system, ultimately resulting in BW [41,42].AhCML69 was highly expressed in the roots, stems, and leaves, indicating that it participated in the entire process of resistance to R. solanacearum in peanut plants.Subcellular localization assays indicated that the AhCML69 protein was localized in both the cytoplasm and nucleus (Fig. 5C).Several CML proteins, such as NbCML30, GhCML11, TaCML36, and CaCML13, have likewise been demonstrated to be located in both the cytoplasm and nucleus and to be involved in the response to biotic stress [15].Silencing of NbCML30 increased tobacco mosaic virus (TMV) infection, while its overexpression inhibited TMV invasion [43].GhCML11 interacts with GhMYB108 to form a positive feedback loop to enhance the defense response against Verticillium dahliae infection in upland cotton [20].Its nuclear-cytoplasmic localization suggested that AhCML69 possibly functions by activating proteins from the nucleus and cytoplasm [44].These results suggested that AhCML69 is involved in the response to pathogens.
As an important oil crop and protein source, peanut plants can be grown in poor-quality soil [45,46], but their yield is significantly restricted by BW caused by R. solanacearum [27].However, studies on the mechanisms of the peanut-R.solanacearum interaction are rare, and no R genes conferring resistance to BW have been identified in peanut.During plant-pathogen interactions, plants activate the two-layer innate immune system, pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), which cause a series of early signaling events, including Ca 2+ flux, mitogen-activated protein kinase (MAPK) activation, reactive oxygen species (ROS) production, the induction of plant hormone biosynthesis, and the hypersensitive response (HR) [47][48][49].As the earliest event, the influx of Ca 2+ from outside plant cells is essential for the immune response [50,51].Ca 2+ signaling is decoded and transduced by sensors that include CML proteins.Accumulating evidence has demonstrated that CMLs are involved in plant protection against pathogen infection [15].In the present study, compared with those in 35S::00 tobacco leaves, leaf necrosis and ROS accumulation were obvious in 35S::AhCML69 tobacco leaves at 72 hpi (Fig. 6A).These findings were consistent with those of two other resistance-related genes identified in previous studies, in which overexpression of AhRRS5 or AhRLK1 in N. benthamiana leaves induced HR and cell death [30,31].Moreover, after 35S::AhCML69 tobacco leaves were inoculated with R. solanacearum, the degree of leaf necrosis was significantly less severe than that in 35S::00 leaves (Fig. 8A).These results demonstrated that the transient overexpression of AhCML69 in tobacco increased its resistance to R. solanacearum infection.
Evidence indicates that CMLs are involved in modulating the transcription of defense-related genes, ultimately causing plant resistance against pathogen infection.As downstream signaling molecules, SA and JA are known plant hormones associated with plant-pathogen interactions [52].AtCML8 and AtCML9 are induced by SA treatment and increase the expression of the SA-dependent PR1 gene in Arabidopsis in response to P. syringae [19,53].AtCML37 and AtCML42 are involved in the defense against herbivory and some pathogen attacks and are associated with calcium and JA signaling [24].In this study, compared with those in the 35S::00 control, the expression of the SA signaling marker genes NbPR1 and NbPR2 and the JA signaling gene NbOPR3 and NbLOX was significantly upregulated in 35S::AhCML69 tobacco leaves infected with R. solanacearum (Fig. 8F, G).Moreover, the expression of the HR marker genes NbH1N1 and NbHsr203J and the PTI marker genes NbWIPK and NbPTI5 was also significantly induced after inoculation of 35S::AhCML69 tobacco leaves with R. solanacearum (Fig. 8D, E).These results were consistent with those elucidated in the study of AhRRS5 and AhRLK1 function, where HR-, JA-and SA-signaling pathway-related genes were all induced and significantly upregulated in tobacco leaves overexpressing the AhRRS5 or AhRLK1 gene [30,31].SA signaling is usually associated with R gene-mediated disease resistance and induces the expression of several CML genes [54][55][56].Although the SA and JA defense pathways are usually antagonistic, synergistic functions have also been found in the defense response to pathogens [57][58][59].These lines of evidence suggest that the AhCML69 gene is involved in the HR, PTI, JA and SA pathways as a positive regulatory factor that modulates resistance to R. solanacearum.

Conclusions
In the present study, 67, 72, and 214 CaM/CML genes were identified in A. duranensis, A. ipaensis, and A. hypogaea, respectively, and were divided into nine subgroups (Groups I-IX).There were 81 pairs of homologous genes in the AhCaM/CML gene family, and the Ka/Ks ratios of these gene pairs were all less than 1.0.The gene duplication events of the AhCaM/CML genes included whole-genome duplication, tandem repeats, and scattered repeats.Expression analysis revealed that AhCML69 was constitutively expressed in the roots, stems, leaves, and flowers of peanut plants and was involved in the response to R. solanacearum infection.The AhCML69 protein was localized in the cytoplasm and nucleus.Transient overexpression of AhCML69 in tobacco leaves increased resistance to R. solanacearum infection and induced the expression of defense-related genes, suggesting that AhCML69 is a positive regulator of PTI by mediating the JA and SA pathways.

Identification of the CaM/CML gene family
The genomes of the cultivated species (A.hypogaea, Shitouqi) and the wild species (A.duranensis and A. ipaensis) were retrieved and downloaded from the corresponding websites [34,60].Seven AtCaM and fifty Arabidopsis AtCML proteins from the TAIR (www.arabi dopsis.org) database were subjected to BLASTP (E value < 1e-5) searches against the protein sequences from the A. hypogaea, A. duranensis and A. ipaensis genome databases.The EF-hand domain was characterized using SMART (http:// smart.embl-heide lberg.de/) and InterPro (http:// www.ebi.ac.uk/ inter pro/).A self-BLAST analysis of the CaM/CML proteins was performed to remove redundant protein sequences with coding sequences less than 1 kb in length, and the remaining sequences were considered putative CaM/CML proteins for further analysis.The molecular weights (MWs) and isoelectric points (pIs) of the CaM/CML proteins were predicted via the Ensembl Genome Browser and ProtComp 9.0 (http:// linux1.softb erry.com/).

Chromosomal localization, gene structure and conserved motif analysis
The length information and location information of the CaM/CML genes were obtained from the GFF3 files on the genome database.The latest TBtools software [61] was employed to analyze the gene structures and chromosomal localization.The conserved motifs analysis of CaM/CML proteins were performed using the MEME program (http:// memee suite.org/ tools/ meme) with the default parameters.

Multiple sequence alignments, phylogenetic analysis and collinearity analysis
Multiple sequence alignments were carried out using the ClustalW tool.A maximum likelihood (ML) tree was constructed by MEGA 11 software based on the full length of the CaM/CML protein sequences to investigate the evolutionary relationship among CaM/CML proteins.The collinearity relationships of the CaM/ CML genes were analyzed by the Multiple Collinearity Scan toolkit MCScanX (http:// chibba.pgml.uga.edu/ mcsca n2/) within the A. hypogaea genome and between homologous proteins in the A. duranensis and A. ipaensis genomes.The results were visualized using TBtools.The Ka/Ks ratios between AhCaM/CML members was calculated by KaKs_Calculator2.0 software [62].

Plant materials and methods
The N. benthamiana seeds were evenly sown in a pot with nutrient soil.After the expansion of the third true leaf, one seedling was transferred into one pot and subsequently grown in a greenhouse at 28 ± 2 ℃ under a 16 h/8 h (light/dark) photoperiod.Four weeks later, these plants were subjected to transient expression assays.Peanut plants were cultivated according to our previous study [63].Different tissues of peanut plants with seven to eight fully grown leaves were used for RNA extraction.

Gene cloning and vector construction
The full CDSs of the AhCML genes were retrieved from our transcript data for peanut cultivars infected with R. solanacearum [33], and the primers for cloning were designed using SnapGene 4.1.8(Additional File 10).The first-strand cDNA of peanut A165 [33], which was infected with R. solanacearum, was chosen as the amplification template for cloning the AhCML genes.The cloned genes were ligated to the PC2300-35S-eGFP vector and transformed into Escherichia coli DH5α.The PCR products of PC2300-35S-AhCML69/AhCML174-eGFP were recovered for gel detection and sent to Guangzhou Qingke Biotechnology Co., Ltd., for sequencing.The verified PC2300-AhCML69/AhCML174-eGFP vectors were subsequently transformed into Agrobacterium tumefaciens GV3101 for transient expression analysis.

Transient expression and R. solanacearum infection
A. tumefaciens cells harboring the PC2300-35S-AhCML69/AhCML174-eGFP construct were grown overnight in LB liquid medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, 1 mL/L kanamycin (50 mg/ mL), 1 mL/L rifampicin (50 mg/mL)) at 28 °C.Bacterial cells were centrifuged and resuspended in infiltration buffer (10 mM MES, 10 mM MgSO 4 , and 200 mM acetosyringone, pH 5.6).The OD 600 of the resuspended liquid was adjusted to 0.1.A. tumefaciens containing the empty vector or the PC2300-35S-AhCML69/AhCML174-eGFP vector was infiltrated into two sides of the same leaf using a 1.0 mL sterile syringe.Two different leaves of one N. benthamiana plant were used.The infiltrated plants were cultured in a greenhouse and subjected to further analysis at different timepoints.R. solanacearum cells were prepared according to the methods of a previous study [63].N. benthamiana was inoculated with R. solanacearum at 48 h after infiltration with A. tumefaciens.

Electrolyte leakage, trypan blue and DAB staining
Six leaves of three N. benthamiana plants were used for the electrolyte leakage measurement, trypan blue staining, and DAB histochemical analysis.Six pieces from every leaf were excised with a puncher of 6 mm diameter for electrolyte leakage measurement.The detailed procedures were described in our previous study [33].For trypan blue, the six leaves were boiled for 2 min in trypan blue solution and incubated overnight in the dark at 28 °C.The stained leaves were destained in chloral hydrate solution (1.25 g/mL) at 25 °C and 50 r/min.The solution was changed every 3 h until the leaves were completely colorless, and photos of the leaves were taken.For DAB staining, the six leaves were subjected to vacuum for 2 min at 0.8 MPa in DAB staining solution and incubated overnight at 28 °C.These leaves were transferred into 90% ethanol to boil until completely colorless.These treated leaves can be preserved in absolute ethanol for a long period of time.

Total RNA extraction and expression profiles
The leaves were collected from transiently transformed N. benthamiana plants or plants inoculated with R. solanacearum at 0, 24, 48, 72 and 96 h after infiltration.Different tissues of peanut plants were sampled from the cultivar Zhongkaihua 1.Total RNA was extracted using a HiPure Plant RNA Mini Kit B (Magen Biotechnology, Guangzhou, China) in accordance with the manufacturer's instructions.The RNA from peanut tissues and N. benthamiana leaves was reverse transcribed to synthesize first-strand cDNA for RT-qPCR according to the instructions of the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotechnology, Beijing, China).RT-qPCR analysis was performed according to the methods of a previous study [33].The immune-related marker genes used in this study included the HR marker genes NbH1N1 and NbHsr203J, the PTI marker genes NbWIPK and NbPTI5, the JA signaling pathway genes NbOPR3 and NbLOX, and the SA signaling pathway genes NbPR1 and NbPR2.NbEF1a and Ahactin were used as the internal reference genes for N. benthamiana and peanut, respectively.The primers used for RT-qPCR are listed in Additional File 10.
The expression data for the representative AhCML genes in different tissues were downloaded from the Peanut Genome Resource (PGR) website (http:// peanu tgr.fafu.edu.cn/).Transcriptome data were obtained from our previous study [33].Heatmaps of the expression profile values of the AhCML genes were generated with TBtools.

Subcellular localization
Transient expression of AhCML69 in N. benthamiana was performed according to the aforementioned protocol.Two days later, the infiltrated leaves were cut and observed under a Leica TCS SP8 confocal laser microscope (Leica Microsystems (Shanghai) Trading Co., Ltd., Mannheim, Germany).A wavelength of 488 nm was used for GFP excitation, and the 510 nm wavelength of the emission signal was obtained for the GFP channel.A wavelength of 640 nm was used for chlorophyll excitation, and 675 nm for the emission signal was used for the chlorophyll channel.

Statistical analysis
Statistical analysis and graphs were performed by using the GraphPad Prism 8.0 software.

Fig. 1
Fig. 1 Phylogenetic and conserved motif analysis of the CaM/CML proteins in A. hypogaea, A. duranensis and A. ipaensis.A Phylogenetic tree of CaM/CML proteins from A. hypogaea, A. duranensis, A. ipaensis, and Arabidopsis was constructed with 1000 bootstrap replications.The different subgroups are distinguished using different colors.B Sequence logo of the six motifs.C The distributions of motifs 1-6 in the AhCaM/CML, AdCaM/ CML, and AiCaM/CML proteins

Fig. 2
Fig. 2 Collinearity analysis of CaM/CML genes in A. hypogaea, A. duranensis and A. ipaensis.A Duplication events in AhCaM/CML genes.The red lines indicate collinear CaM/CML gene pairs in A. hypogaea.B Collinearity analysis of the CaM/CML genes in A. hypogaea, A. duranensis and A. ipaensis.Homologous CaM/CML gene pairs between species are linked by yellow and red lines, respectively

Fig. 3
Fig. 3 Heatmap of the expression profiles of the AhCaM/CML genes and expression analysis of AhCML69.A Expression profiles (in log10-based FPKM) of the representative AhCaM/CML genes from 29 peanut tissues.The expression abundance of each gene is represented by the color bar: red indicates higher expression, and green indicates lower expression.FPKM, Fragments Per Kilobase of Transcript per Million mapped reads.B Expression profiles (in log10-based FPKM) of the representative AhCaM/CML genes in the leaves of resistant and susceptible peanut plants at 0, 24, 36, 48 and 72 h post-inoculation with R. solanacearum.C Expression analysis of AhCML69 in different peanut tissues.Error bars represent standard error, n=3

Fig. 4
Fig. 4 Functional analysis of the representative AhCML genes.A Structure of the transient overexpression vectors containing AhCML69 and AhCML174.B Phenotypic analysis of tobacco leaves agroinfiltrated with Agrobacterium in the control (35S::00) and experimental (35S::AhCML69 and 35S::AhCML174) groups at 48, 72, 96, 120 and 144 h.The numbers indicate the number of agroinfiltrated leaves on the bottom

Fig. 5
Fig. 5 Sequence analysis and subcellular localization of AhCML69.A Multiple sequence alignment of the CML44 proteins from Arabidopsis, rice, pepper, and A. hypogaea.B Phylogenetic tree of the CML44 proteins in Arabidopsis, rice, pepper, and A. hypogaea.C Subcellular localization of the AhCML69 protein

Fig. 8
Fig. 8 Analysis of the function of AhCML69 in resistance to R. solanacearum in N. benthamiana leaves.A Phenotypic analysis of the tobacco leaves after inoculation with R. solanacearum for 24 and 48 h (from left to right).Cell death was monitored by visible light (Camera) and UV light (UV); bar = 5 cm.B The expression levels of AhCML69 in tobacco leaves agroinfiltrated with Agrobacterium containing 35S::00 or 35S::AhCML69 at 24 and 48 h.Different lowercase letters indicate significant differences according to ANOVA (mean ± SE, p < 0.05).C Electrolyte leakage in tobacco leaves agroinfiltrated with Agrobacterium expressing 35S::00 or 35S::AhCML69.Different lowercase letters indicate significant differences according to ANOVA (mean ± SE, p < 0.05).D-G Expression analysis of immune marker genes in tobacco leaves agroinfiltrated with 35S::00 and 35S::AhCML69 at 48, 72 and 96 h.Different lowercase letters indicate significant differences according to ANOVA (means ± SEs, p < 0.05)